AbstractSoil microorganisms coexist and interact showing antagonistic or mutualistic behaviors. Here, we show that an environmental strain of Bacillus subtilis undergoes heritable phenotypic variation upon interaction with the soil fungal pathogen Setophoma terrestris (ST). Metabolomics analysis revealed differential profiles in B. subtilis before (pre-ST) and after (post-ST) interacting with the fungus, which paradoxically involved the absence of lipopeptides surfactin and plipastatin and yet acquisition of antifungal activity in post-ST variants. The profile of volatile compounds showed that 2-heptanone and 2-octanone were the most discriminating metabolites present at higher concentrations in post-ST during the interaction process. Both ketones showed strong antifungal activity, which was lost with the addition of exogenous surfactin. Whole-genome analyses indicate that mutations in ComQPXA quorum-sensing system, constituted the genetic bases of post-ST conversion, which rewired B. subtilis metabolism towards the depletion of surfactins and the production of antifungal compounds during its antagonistic interaction with S. terrestris.